A. Field of Invention
The present invention relates generally to the pattering of cell culture surfaces to maximize the ability of such surfaces to support adhesion, growth, colonization and differentiation of selectable cell lines.
B. Prior Art
Surface microgeometry plays a significant role in in vitro cell surface interactions as well as in in vivo tissue-implant surface interactions, although well-defined casual relationships are not yet fully established. For example, defined surface microgeometries such as grooved and machined metallic and polymer surfaces cause orientation of cells and matrix molecules thereof in vivo. See Chehroudi, et al xe2x80x9cTitanium-coated micromachined grooves of different dimensions affect epithelial and connective tissue cells differentially in vivo,xe2x80x9d Journal of BioMedical Materials Research, 24:1203-1219 (1990), Matsuda, et al xe2x80x9cDevelopment of micropattering technology for cultured cells,xe2x80x9d Transactions of the American Society For Artificial Internal Organs, 36:M559-M562 (1990); Clark, et al, Topographical Control of Cell Behavior II. Multiple Grooved Substrates, Development, 108 (4) 1990; and Clark, et al, Cell Guidance by Ultra Fine Topography in vitro, Journal of Cell Science, 99 Part 1 (March 1991). It is further known that laser-microgrooved titanium surfaces can promote organized bone formation and integration around implants, and that surface texturing also enhances platelet attachment as well as fibrin clot adhesion, thereby improving the stability of implant tissue interface during colagenous matrix formation and contractrue during healing.
It is believed that surface texturing operates on multiple levels to improve implant stability and adhesion this, inter alia, due to the fact that textured surfaces have larger surface areas than do smooth surfaces, thus creating larger more stable mechanical interfaces. As such, textured surfaces also promote adhesion of fibrin and other, more permanent cell matrix components, and thus affect long term cell interactions at stable interfaces. In the short term, that is, soon after implantation, fibrous tissue cells are more organized and collagenous at smooth interfaces than at textured interfaces. This, of course, is not desirable given that fibrous cells constitute a principal component of scar tissue. In distinction, textured surfaces have the additional advantage over smooth surfaces of inhibiting colonization of fibrous tissue cells, also known as fibroblasts and of macrophagic cells which appear early during wound healing and normally encapsulate smooth substrates. It has also been recently shown that textured surfaces may also promote the differentiation of bone forming (oseogenic) cells which form at critical interfaces with implant surfaces.
More generically, and with particular reference to the subject matter of the instant invention, cell culture technology constitutes an essential tool for bio-materials and bio-medical engineering research as well as for basic science and bio-technical research. The controlled nature of the culture environment enables investigators to study cultured cells as experimental models, that is, microcosms, of actual living tissues. This technology is largely based on the propagation and analyzing of cells attached to culture substrates. However, most culturing occurs upon flat surfaces which, it has been demonstrated, promote rapid spreading of cells, uncontrolled cell growth, and the loss of cell differentiation, all of which impact upon the value of the culture environment as a tool of basic science and biotechnology research. There is therefore a need in the art for advanced culture surfaces that can be employed to control cell attachment, spreading, proliferation, and differentiation. The instant invention furthers these ends through the use of discrete surface microstructures based upon characteristics which are believed to be inherent to cell function and structure itself.
The invention is thereby particularly intended to provide a bio-reactor or cell culture surface which is more design specific for purposes of control of cell attachment, contact guidance, and cell shape, to a much higher degree than that in simple microgrooving of surfaces heretofore known in the art. The present invention is also significant in terms of modulation of the behavior of cultured mesenchymal stem cells as well as of differentiated connective tissue cell lines.
The prior art, as it relates to issued patents relative to cell culture surfaces, is reflected by U.S. Pat. No. 5,202,227 (1993) to Matsuda, et al, entitled Control of Cell Arrangement; and U.S. Pat. No. 5,976,826 (1999) to Singhvi, et al, entitled Device Containing Cytophilic Islands That Adhere Cells Separated By Cytiphobic Regions.
A bioreactor or cell culture surface comprises a multiplicity of microwells appearing, upon enlargement in the range of 200 to 1000 magnifications, as a honeycomb structure consisting of a plurality of rows of irregular hexagons, each having a transverse x-axis, a longitudinal y-axis, and a z-axis which defines a height of each microwell. Co-parallel major bases define the directionality of anticipated cell growth, while pairs of equilateral minor bases define the x-axis width of each microwell. An arrow-like geometry is defined by said equilateral pairs of minor bases which also define an x-axis separation between opposing major bases of the hexagon to induce cell growth directionality. An acute angle exists between the members of said arrow-like pairs of minor bases, while an obtuse angle exists between contiguous major and minor bases. The irregular hexagonal geometry also contributes to a convenient offset between x-axis rows of microwells relative to each other, so that tagging and observation thereof is simplified, and so that the potential for overgrowth of a cell or cell colony within a given microwell to an adjoining microwell of a different x-axis column of the cell culture surface is diminished. The depth of each microwell wall is preferably four micrometers, the x-axis width thereof in a range of 5 to 15 micrometers, and the y-axis length thereof in a range of 1 to 8 times said value of the width. The ratio of length to width, that is, the y/x aspect ratio, is typically in a range of 1:1 to 8:1 in which the thickness between adjacent major bases of adjacent microwells within a single x-axis column is one micrometer. Such cell culture surfaces can be formed using state of the art techniques such a photolithography, in etching, or polymer fabrication technology such as molding.
It is accordingly an object of the invention to provide an improved bioreaction and cell culture surface having microgeometric surface features specific to the modulation of cell growth and differentiation.
It is another object to provide a bioreactor of the above type capable of defining important cell dimension parameters for purposes of surface micrortextuing of anticipated orthopedic and other prosthetic devices.
It is a further object of the invention to provide an improved tool for the basic sciences associated with cellular research in terms of cell functions, inclusive of control of cell attachment, spreading, orientation, differentiation, and gene expression.
It is a yet further object to provide a bioreactor having a tissue-biomaterial interface permitting the fullest expression of mammalian cell types and their characteristics.
It is a still further object of the invention to provide a micro-formed substrate having particular utility as a cell culture tool and, more particularly, utility in the modulation of cultured mesenchymal stem cells and differentiated connective tissue cells.
It is another object to furnish a cell culture surface having particular use the study of the attachment, proliferation and differentiation of osteoblast-like cells.
It is another object of the invention to provide a bio-reactive surface of the above type that may be used to optimize design parameters for surfaces of implantable medical devices such as dental implants and total joint replacements.
It is a yet further object to provide a surface to provide a new and improved tool of basic research in cell biology.
It is a still further object of the invention to provide a cell culture surface of the above type having a surface chemistry to optimize ability of bio-medical substrates thereof to support the growth of osteogenic cells.